Dapi containing mounting medium

Cells were seeded onto glass coverslips in 24-well plates and incubated for 24 h until about 70% confluence. Cells were washed three times with ice-cold PBS, fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature (RT), permeabilized with 0.3% Triton X-100 for 30 min, and blocked with 10% goat serum for 40 min, followed by incubation with primary antibodies (1 h) and fluorescence-labeled secondary antibodies (1 h). Slides were sealed within DAPI-containing mounting medium (SouthernBiotech, 0100–20). We note that for labeling endogenous TM9SF2, cells were fixed in methanol at −20 °C for 10 min, as this antibody did not work on samples fixed by PFA. For the toxin surface binding assay, cells were incubated with toxin-containing medium (4.8 μg/mL Stx1, or 2 ng/mL Ctx-555) on ice for 60 min. Cells were washed, fixed, and subjected to immunostaining without permeabilization. Stx1 polyclonal antibody was used to recognize surface-bound Stx1. Fluorescent images were captured with the Olympus DSU-IX81 Spinning Disk Confocal System. Images were pseudocolored and analyzed using ImageJ.

Cells were seeded in 4-well chamber slides, and then serum-starved overnight in DMEM containing 0.1% FBS. A scratch assay was performed as described above. After cell migration occurred overnight, cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100, blocked with 2% Bovine Serum Albumin + 0.3% Triton X-100, and incubated with anti-paxillin primary antibody (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. After that, cells were incubated with Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 hour. Three washes with PBS were performed in-between steps. Cells were then mounted with a DAPI-containing mounting medium (Southern Biotech, Birmingham, AL, USA). Five images of pre-selected areas at the edge of the scratch (kept consistent between groups) were taken with the same camera settings for all treatment groups using a fluorescence microscope and camera (model BX51 and DP70, Olympus, Shinjuku, Tokyo, Japan). After setting the same particle size threshold for all the groups, particles were automatically counted within the cells in each picture using the software ImageJ and normalized by the number of DAPI-stained nuclei in the same picture. Results are shown as fold change vs. the negative control group.

Cells were seeded on 8-well chamber slides at a density of 4,000–6,000 cells/well in DMEM containing 10% FBS for 24 hours, then serum-starved overnight in DMEM containing 0.1% FBS. Cells were pre-treated with AT-RvD1 (10nM) or vehicle (ethanol) with or without Rp-8-Br-cAMP (10μM) for 2 hours, followed by the addition of PDGF-BB (10ng/ml) for 1 hour. Cells were then fixed in 3.7% formaldehyde, permeabilized in 0.1% Triton X-100, stained with Alexa Fluor 568 phalloidin and mounted with a DAPI-containing mounting medium (Southern Biotech, Birmingham, AL, USA). One wash with PBS was performed in-between steps. Images (10x) from five pre-selected areas (consistent between groups) per well were taken using a fluorescence microscope and camera (model BX51 and DP70, Olympus, Shinjuku, Tokyo, Japan), and length to width ratios were measured for each cell (≥20 per well) using the software ImageJ.

Frozen samples were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton‐X‐100, blocked with 5% foetal bovine serum (Gibco, Cat No. 10099141C) and incubated with diluted (1:200) fluorochrome‐conjugated antibodies against mouse Ly‐6G (BD Biosciences, Cat No.551461), CD41 (Biolegend, Cat No.133913) or HDC (Abcam, Cat No. ab37291) overnight at 4°C. The nuclei were stained with DAPI‐containing mounting medium (Southern Biotech, Cat No.0100‐20). Images were acquired using a Leica SP8 confocal microscope and processed with the LASX software.

iPSCs or iPSC-derived neurons were cultured as described above, then washed three times in PBS before fixation in 4% paraformaldehyde for 10 min. Cells were washed in PBS, then permeabilised in 0.2% Triton X-100 in PBS for 5 min. After further washing, cells were incubated in blocking buffer (10% donkey serum in PBS) in PBS for 3 h at room temperature followed by incubation with primary antibody (overnight, 4 °C). Cells were then washed, incubated with secondary antibody (conjugated to Alexa Fluor 488 (RRID: AB_2556542) or Alexa Fluor 568 (AB_25340); Life Technologies, Paisley, UK), followed by washing in PBS and mounting on slides using DAPI-containing mounting medium (Southern Biotech, Birmingham, AL, USA). Slides were visualised using the Evos FL (Thermo Fisher Scientific, Loughborough, UK). Primary antibodies used were for: Sox2 (AB_2341193), SSEA4 (AB_778073), Oct4 (AB_445175), Nanog (AB_446437), MAP2 (AB_297885), βIII tubulin (AB_444319), Tbr1 (AB_2200219) and Sat2b (AB_882455) (all Abcam, Cambridge, UK).